M148R and M149R are two virulence factors for myxoma virus pathogenesis in the European rabbit

Vet. Res. (2009) 40:11 www.vetres.orgDOI: 10.1051/vetres:2008049

C© INRA, EDP Sciences, 2009 Original article

M148R and M149R are two virulence factors for myxomavirus pathogenesis in the European rabbit

Sophie Blanié1,2, Jérémy Mortier1,2, Maxence Delverdier1,2,Stéphane Bertagnoli1,2, Christelle Camus-Bouclainville1,2*

1 INRA, UMR 1225, F-31076 Toulouse, France2 Université de Toulouse ; ENVT ; UMR 1225 ; F-31076 Toulouse, France

(Received 25 July 2008; accepted 13 November 2008)

Abstract – Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible formyxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). MYXV has a lineardouble-stranded DNA genome that encodes several factors important for evasion from the host immunesystem. Among them, four ankyrin (ANK) repeat proteins were identified: M148R, M149R, M150R andM-T5. To date, only M150R and M-T5 were studied and characterized as critical virulence factors. Thisarticle presents the first characterization of M148R and M149R. Green Fluorescent Protein (GFP) fusionsallowed us to localize them in a viral context. Whereas M149R is only cytoplasmic, interestingly, M148Ris in part located in the nucleolus, a unique feature for an ANK repeat poxviral protein. In order to evaluatetheir implication in viral pathogenicity, targeted M148R, M149R, or both deletions were constructed inthe wild type T1 strain of myxoma virus. In vitro infection of rabbit and primate cultured cells as well asprimary rabbit cells allowed us to conclude that M148R and M149R are not likely to be implicated in celltropism or host range functions. However, in vivo experiments revealed that they are virulence factors sinceafter infection of European rabbits with mutant viruses, a delay in the onset of clinical signs, an increaseof survival time and a dramatic decrease in mortality rate were observed. Moreover, histological analysissuggests that M148R plays a role in the subversion of host inflammatory response by MYXV.

poxvirus / myxoma virus / ankyrin repeat / virulence / rabbit

1. INTRODUCTION

Myxoma virus (MYXV), a member of thePoxviridae family, is the agent responsiblefor myxomatosis, a highly lethal disease inthe European rabbit (Oryctolagus cuniculus).The main anatomical and clinical features ofmyxomatosis are a pseudotumoral lesion at theinoculation site followed by the occurrence ofsecondary lesions at cutaneous and viscerallevels, named myxomas [29]. Due to MYXV’sparticular ability to escape and subvert thehost artillery, these lesions are accompaniedby general dysfunction of cellular immunity,resulting in bacterial super infections of therespiratory tract culminating in death withintwo weeks [29].

* Corresponding author: [email protected]

MYXV has a double-stranded DNAgenome of 162 kbp [5], with a central regioncontaining highly conserved enzymatic andstructural genes required for the maintenanceof essential viral functions. Peripheral regionsof the DNA, within and near the invertedterminal repeats (ITR) at both sides of thegenome, encode nonessential factors that con-tribute to the modulation of the host responseto infection [29, 31, 32]. Two genes presentnear the right ITR, M148R and M149R,have functions that are still speculative. Theyencode 2 of the 4 ankyrin (ANK) repeat-containing proteins of the MYXV [5, 17].The ANK repeat is one of the most common,modular, protein-protein interaction motifsin nature. This module is involved in a widerange of cellular functions. The importance

Vet. Res. (2009) 40:11 S. Blanié et al.

of the MYXV-ANK-repeat proteins has beendemonstrated: MT-5 is a host-range factoressential for permissive MYXV infection inrabbit lymphocytes [18, 34], and MyxomaNuclear factor (MNF) seems to interfere withNF-kappa B (NF�B) pathways, leading tothe inhibition of inflammatory response [6].Disruption of either one of these genes resultsin dramatic attenuation of myxomatosis ininfected European rabbits. Interestingly,M148R, M149R, and MNF (M150R) arelocated in series on the MYXV genome,forming a cluster of ANK repeats encodinggenes at the right end of the MYXV genome.

Here we present the first characterizationof M148R and M149R. Our data suggest thatthey are both virulence factors of MYXV, withdifferent cellular localizations and acting indifferent ways compared to the other ANKrepeat MYXV proteins.

2. MATERIALS AND METHODS

2.1. Cells and viruses

Rabbit kidney cells (RK13; ATCC CCL-37),RK13 HGPRT− cells (deficient in hypoxanthine-guanine phosphoribosyl transferase) and BabyGreen Monkey Kidney cells (BGMK) were main-tained in Dulbecco minimum essential medium(DMEM) supplemented with 10% fetal calf serum(FCS). Rabbit peripheral blood mononuclear cells(PBMC) were isolated and cultured as previouslydescribed [23].The wild type MYXV strain T1 andthe MYXV mutants (MYXV-�M148R, MYXV-�M149R and MYXV-�M148R�M149R) weregrown in RK13 cells, in DMEM supplemented with5% FCS.

2.2. Cloning, sequencing and computer analysisof DNA and protein sequences

DNA sequences were analyzed with DNAStrider 1.3 software [15] and the BLAST pro-gram [35]. Searches for ANK repeats wereperformed with REP1 [1]; Pfam2 [11]; andSMART3 [14]; online software packages. Manual

1 http://www.embl-heidelberg.de/∼andrade/papers/rep/search.html2 http://pfam.sanger.ac.uk/search3 http://smart.embl-heidelberg.de/

analysis, according to published consensus pro-moter sequences [4], was performed to predictM148R and M149R expression during the infectioncycle.

2.3. RNA extraction and RT-PCR analysis

RK13 cells (2 × 106 cells) were infected withwild type MYXV strain T1 at a multiplicity ofinfection (m.o.i.) of 5. The inoculum was allowedto adsorb for 1 h at 4 ◦C. Total RNA was isolated at0, 2, 4, 8, 12 and 16 h post-infection (p.i.) (Nucleo-Spin RNA II; Macherey-Nagel, Hoerd, France). Inaddition, total RNA was also extracted at 16 h p.i.from mock infected cells and from T1 infected cellstreated with 40 �g/mL cytosine arabinoside (AraC)(Sigma, Lyon, France) at the time of infection. AllRNA samples were subjected to DNaseI treatment.Reverse transcription was performed using oligo-dT and M-MLV reverse transcriptase (Invitrogen,Paisley, UK), and followed by PCR analysisusing specific primers of M148R (5′M148R-BglII(5′-CGAAGATCTATGGATCACGTAAGCTATTACATTGT-3′) and 3′M148R-PstI (5′-AAACTGCAGGTAACCGAACGTATCTATGCAAC-3′))or M149R (5′M149R-BamHI (5′-CGC GGATCCGCAATTATGGCCTCGTTGCAT-3′) and 3′M149R-PstI (5′-AAACTGCAGCGCACTATTTTGTACGGTTTA-3′) (restriction sites are underlined).PCR (Expand High Fidelity; Roche, Basel,Switzerland) were performed over 30 cycles, with ahybridization temperature of 63 ◦C for each cycle.A control RT-PCR, in which reverse transcriptasewas omitted during the RT step, was performedwith all samples (not shown).

2.4. Engineering of Green Fluorescent Protein(GFP) fusions

GFP fusions, under CMV promoter, wereobtained using the pEGFP-F plasmid vector (BDBiosciences-Clontech, Saint-Germain-en-Laye,France). The M148R open reading frame wasamplified by PCR using 5′M148RBglII and3′M148RPstI. The M149R open reading framewas amplified by PCR using 5′M149RBamHIand 3′M149RPstI. The resulting PCR fragmentswere inserted between the BglII and PstI sitesin pEGFP-F, thus replacing the farsenylationbox-coding sequence downstream of the GFPsequence. The resulting plasmids have been namedpCMV-GFPM148R and pCMV-GFPM149R.

Page 2 of 14 (page number not for citation purpose)

M148R and M149R are MYXV’s virulence factors Vet. Res. (2009) 40:11

The P7.5 early/late poxviral promoter, issuedfrom pSC11 [7], was digested by XbaI-BamHIand cloned into pBluescript-SK (Stratagene, Massy,France). The resulting plasmid pBS-P7.5 wasdigested by XmaI and KpnI to insert, underP7.5 poxviral promoter, the entire GFP fusionsequence of M148R or M149R, provided byAgeI-KpnI digestion of pCMV-GFPM148R andpCMV-GFPM149R, respectively. The resultingplasmids are pBS-P7.5GFPM148R and pBS-P7.5GFPM149R, respectively.

2.5. Confocal microscopy observations

BGMK cells were plated onto multichamberslide flasks (Falcon, Le Pont de Claix, France),transfected with plasmid expressing GFP fusionof M148R or M149R under P7.5 or CMVpromoter, with or without prior infection for2 h with MYXV deleted for the correspondinggene. Twenty-four hours post-transfection (p.t.),cells were fixed with 4% paraformaldehyde inPBS and permeabilized with 0.1% Triton X-100. Cells were incubated with mouse anti-humanB23 antibodies (1/100; Sigma) or anti-MYXVserum (1/500) in 0.05% Tween in PBS and thenwith rhodamine RX coupled anti-mouse antibod-ies (1/200; Jackson Immunoresearch, West Grove,PA, USA) or CY-5 coupled anti-rabbit antibodies(1/200; Immunotech, Villepinte, France), respec-tively. Some samples were stained with TO-PRO-3iodide (Invitrogen). Samples were observed with aconfocal LSM Olympus microscope. Multistainingexperiments were performed using the sequentialmode.

2.6. Construction of MYXV-�M148R,MYXV-�M149R,MYXV-�M148R�M149R,and revertant viruses

Briefly, inactivation of the target open readingframes (M148R, M149R or both) was achievedby homologous recombination in MYXV infectedRK13 cells, transfected with the appropriateplasmid. The M149R gene, amplified by PCR(using 5′M149RBamHI and 3′M149RPstI primers)and cloned into the pGEM-T vector (Promega,Charbonnieres, France) was inactivated by replac-ing the SnaBI-BglII central region (785 bp) by theLacZ gene, under the control of the late Vaccinia

virus promoter P11, issued from pSC11 [7]. Afterhomologous recombination in MYXV T1 infectedcells, MYXV-�M149R mutant was screened forblue plaque in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-�-d-galactopyranoside).

M148R gene, amplified by PCR (using5′M148RBglII and 3′M148RPstI ) and cloned intothe pGEM-T expression vector (Promega), wasinactivated by replacing the SnaBI-EcoRV centralregion (1051 bp) by the Ecogpt gene (Escherichiacoli xanthine-guanine phosphoribosyl transferase),under the control of the P7.5 promoter issued fromthe digest of pRBgpt [9]. After recombination inMYXV infected RK13 cells, MYXV-�M148Rmutants were selected by resistance to mycophe-nolic acid in DMEM supplemented with 25 �g/mLmycophenolic acid (MPA), 250 �g/mL xanthineand HAT (Hypoxanthine Aminopterin Thymidine;Sigma, 1/50).

The MYXV-�M148R�M149R was obtainedby transfecting MYXV-�M149R infected RK13cells with the plasmid containing the inactivatedform of M148R. MYXV-�M148R�M149R wasselected by resistance to mycophenolic acid.Purity of all mutant viruses was verified byPCR.

In order to engineer a revertant virus (MYXV-rev), the native sequence including M148R andM149R open reading frames was obtained by PCRusing 5′M148RBglII and 3′M149RPstI primers,and cloned into pGEM-T. After recombina-tion in MYXV-�M148R�M149R-infected RK13HGPRT− cells, MYXV-rev was obtained by reverseEcogpt selection using 6-thioguanine (5 �g/mL)and by reverse white-blue screening.

2.7. Infection and virus growth curves

For growth analysis in rabbit PBMC, 6 × 106

cells were infected with T1, MYXV-�M148R,MYXV-�M149R or MYXV-�M148R�M149R atan m.o.i. of 1. For single-step analysis on RK13and BGMK, 5 × 105 cells were infected with wildtype MYXV or mutant viruses at an m.o.i. of 4. Formulti-step analysis on RK13 and BGMK, 5 × 105

cells were infected with viruses at an m.o.i. of 0.01.The inoculums were allowed to adsorb for 2 h at

4 ◦C. Then, unabsorbed viruses were removed, andcells were incubated in DMEM supplemented with5% FCS. At different times p.i., cells were scraped,frozen and virus titers were determined on RK13cells.

(page number not for citation purpose) Page 3 of 14


2.8. Infection of rabbits with wild-type MYXV,MYXV-rev, MYXV-�M148R,MYXV-�M149R andMYXV-�M148R�M149R

Eight-week old male New Zealand Whiterabbits were obtained from a local supplier andhoused in biocontainment facilities according tothe guidelines of the European Community Councilon Animal Care4. All procedures on animalswere performed by staff accredited by the FrenchMinistry of Agriculture and were designed to limitanimal pain and distress. Infections were performedintradermally in the right ear with 5 × 103 PFUof either virus. Rabbits were monitored daily forclinical signs of myxomatosis. Rabbits that becamemoribund were sacrificed with T61 (Distrivet,Romainville, France) administered intravenously.

2.9. Histological examination

For histological studies, 6 rabbits were inocu-lated with MYXV strain T1 and 6 were inocu-lated with each mutant-virus described above. At 5,8, and 13 days p.i., two animals from each groupwere sacrificed. Two mock-infected rabbits weresacrificed and used as controls. All animals weresubjected to complete post-mortem examination.Tissue material from the injection site (ear [pri-mary site]) and parotid lymph node (secondary site)were taken and stored in 10% neutral formalin forfurther analysis. After fixation, tissues were rou-tinely processed into paraffin blocks, sectioned at athickness of 4 �m, and stained with haematoxylinand eosin for microscopic examination. Lesionswere assessed histologically and graded as minimal(+), mild (++), moderate (+++), marked (++++) orsevere (+++++).

3. RESULTS

3.1. M148R and M149R are two ANK repeatsand F-box containing proteins

Depending on the software used for proteinanalysis, up to 10 ANK repeats can beidentified in M148R, and up to 9 in M149R(Fig. 1). The newly established PRANC(Pox protein Repeats of Ankyrin C-terminal)

4 European Council directive 86/609/EEC, 24November 1986.

domain, belonging to the F-box motif family,is present in both proteins.

3.2. M148R and M149R are expressedsequentially during the infection

Manual analysis, according to publishedconsensus promoter and terminator sequences,revealed sequences consistent with a latetranscription of M148R. No sequence clearlycorresponding to early or late promoter wasfound upstream of the ATG of M149R.

RT-PCR analysis of total RNA purifiedfrom wild-type MYXV-infected cells demon-strated that the M148R transcript was weaklydetectable as early as 2 h p.i.. The expres-sion of M148R increased up to 16 h p.i. andis totally inhibited in the presence of AraC(Fig. 2, upper panel).

The M149R transcript was detectable asearly as 2 h p.i., and could still be detectedat 16 h p.i. (Fig. 2, middle panel). However,the level of detected transcript was noticeablylower following the treatment with AraC, asopposed to what was observed with MNF(M150R), control of early gene expression(Fig. 2, lower panel). Since M148R, M149Rand MNF are organized in a cluster ofgenes, temporally expressed in the reversesense of their location, one could influencethe expression of the others. Therefore, RT-PCR analysis of expression of M148R andM149R in T1�MNF-infected RK13 cellswas performed. MNF deletion affects neitherM148R nor M149R transcription (data notshown). Likewise, deletion of M149R does notaffect M148R transcription.

3.3. M148R and M149R have differentsubcellular localizations

To determine the subcellular localiza-tion of each protein, plasmids expressing aGFP-protein fusion (with M148R or M149Rfused downstream of the GFP, under CMVpromoter) were used to transfect BGMK cells.Cells were examined by confocal microscopyat 24 h p.t.. As shown in Figure 3Aa, GFP-M148R fusion is located in both the cytoplasmand nucleus. The nuclear fraction of M148R



BM148R

M149R

Figure 1. Functional domains of M148R and M149R Myxoma virus proteins. (A) Functional domains ofM148R and M149R proteins found by 3 online software: Pfam [11], REP [1] and SMART [14]. “trunc”(truncated) indicates that only the second half of the repeat was identified. (B) Schematic representation ofM148R and M149R proteins. Gray boxes: ANK repeat; hatched: PRANC (Pox protein Repeats of AnkyrinC-terminal) domain.

+ - T0 T2 T4 T8 T12 T16 n.i AraC

M148R

M150R

M149R

Figure 2. Expression of M148R and M149R mRNA in cell culture. RK13 cells were infected with wildtype MYXV. At different times p.i., mRNA were extracted, subjected to reverse transcription using oligodTprimers, following which, PCR was performed using specific primers for M148R, M149R, or M150R. Lane+, positive control, i.e. PCR on T1 DNA; lane –, negative control, i.e. PCR without template; lanes T0

through T16, cells infected with wild-type MYXV for 0, 2, 4, 8, 12, or 16 h, respectively; lane n.i., mock-infected cells; lane AraC, cells infected with wild-type MYXV for 16 h and incubated with AraC at the timeof infection. T0 corresponds to RNA extractions performed after 1 h of virus adsorption.



GFP Anti-B23 MERGE

GFP Anti-MYXV MERGE

pCMV-GFP-M148R

pCMV-GFP-M149R

pBS-P7.5GFP-M148R

pBS-P7.5GFP-M149R

A

B

a b c

d e f

a b c

d e f

Figure 3. Cellular localization of M148R and M149R. (A) BGMK cells grown on coverslips weretransfected with pCMV-GFPM148R (a, b, c) or pCMV-GFPM149R (d, e, f). Cells were fixed at 24 h p.t.,permeabilized, stained with anti-B23 antibody (red staining) and TO-PRO-3 iodide to detect nuclei (bluestaining), and analyzed by confocal microscopy with a 60× objective. Multistaining experiments wereperformed using the sequential mode. (B) BGMK cells grown on coverslips were pBS-P7.5GFPM148R(a, b, c) or pBS-P7.5GFPM149R (d, e, f) transfected, with prior 2 h infection with the MYXV deleted forthe corresponding gene. At 24 h p.t., cells were fixed, permeabilized, stained with anti-MYXV serum (redstaining) and analyzed by confocal microscopy as previously indicated.

shows a nucleolar pattern, as confirmed bycolocalization (Fig. 3Ac, yellow merge stain-ing) with B23 (nucleophosmin, Fig. 3Ab, redstaining), a major multifunctional nucleolar

phosphoprotein. A punctuate pattern witha uniform distribution in the cytoplasmwas observed with the GFP-M149R fusion(Fig. 3Ad). It was not possible to show any



colocalization with the endoplasmic retic-ulum, early endosomes, or lysosome (datanot shown). Typical diffuse (cytoplasmic andnuclear) green pattern was observed with thecontrol transfection with a GFP expressingplasmid (data not shown).

The same patterns were observed inan infectious context, with GFP-fusionsexpressed under P7.5 poxviral promoter(Fig. 3B). Absence of green staining in BGMKtransfected with pBS-7.5GFPM148R or pBS-7.5GFPM149R without infectious contextconfirmed that fusion expression under theP7.5 promoter requires MYXV infection (datanot shown).

3.4. M148R and M149R are not essential forreplication in classical MYXV permissivecells

The role of M148R and M149R inthe replication of MYXV was evaluatedby engineering mutant viruses deletedin either M148R (MYXV-�M148R),or M149R (MYXV-�M149R), or both(MYXV-�M148R�M149R). The capacity ofreplication of mutant viruses in cultured celllines (RK13 and BGMK) and primary cells(PBMC) was tested. Experiments showed thatneither deletion of M148R, nor M149R, norboth, induced modification in virus replicationin RK13, BGMK, or PBMC (Fig. 4A). Inorder to examine mutant viruses spread incultured cells, multi-step experiments onRK13 and BGMK cells were performed. Nodefects in the ability of mutant viruses tospread in these cells were observed (Fig. 4B).

3.5. M148R and M149R are new MYXVvirulence factors in European rabbits

To evaluate the role of M148R andM149R in the pathobiology of MYXV,European rabbits were inoculated intrader-mally with either wild type MYXV strainT1, MYXV-�M148R, MYXV-�M149R,MYXV-�M148R�M149R, or MYXV-rev(control of phenotype specificity). The clinicalcourse of infection was monitored daily for21 days. Data are summarized in Table I.Rabbits infected with the T1 strain developed

typical clinical myxomatosis, characterizedby the development at day 4 p.i. of a large redand raised primary skin lesion at the inocu-lation site. At day 8 p.i., secondary lesionsappeared on ears, eyelids, nose, back andsubsequentially over the entire body. Conjunc-tivas inflammation and initially serous, latermucopurulent (day 10 p.i.) discharge fromthe nose and eyes followed, accompanied byrespiratory distress (day 12 p.i.). Rabbits diedwithin two weeks due to bacterial infection ofthe respiratory tract. The same clinical courseof infection was observed with MYXV-revinfected rabbits.

Clinical observations revealed that infectionwith the MYXV-�M148R, MYXV-�M149Rand MYXV-�M148R�M149R mutants isclearly attenuated compared to wild typevirus infection. The MYXV-�M148R infectedgroup showed a delay of three days inthe onset of clinical signs, with a mortalityrate of 40%. They suffered from only lightinfections of conjunctivas and respiratorytract. MYXV-�M149R induced a delay of4 days on the onset of secondary lesions,which were less numerous and restricted tothe face area. All infected animals recovered.Finally, all rabbits infected with the MYXV-�M148R�M149R showed few clinical signs,except for secondary lesions which appearedearlier (day 6 p.i.) than usual, were smaller andmore numerous, and finally resorbed rapidly.All rabbits recovered.

3.6. Histological analysis of lesions from wildtype and mutant MYXV

Detailed histological examination of tissuematerial from both the primary (ear) andsecondary (parotid lymph node) infection siteswas carried out at various times during thecourse of infection. The results of the completeanalysis are summarized in Table II.

At day 5 p.i., the injection sites of wild-typeMYXV and mutant viruses demonstrated sim-ilar microscopic lesions, i.e. perivascular der-matitis with focal edema, interstitial mucinosisand inflammatory infiltrates mainly composedof heterophils. In the parotid lymph node,both mutant and wild-type viruses induced a



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107

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104

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107

105

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104

107

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Figure 4. Growth kinetics of wild-type and mutant viruses. (A) To evaluate viruses in vitro replication, celllines (RK13 and BGMK) and primary cells (PBMC) were respectively infected at m.o.i. 4 and m.o.i. 1,with wild-type MYXV (thin solid line (�)), MYXV-�M148R (bold dashed line (�)), MYXV-�M149R(thin dashed line (�)) or MYXV-�M148R�M149R (bold solid line (×)). Virus titers were determined atdifferent times p.i. on RK13 cells. Results are shown as means ± SEM (triplicate). (B) To examine virusspread, cells were infected at a low m.o.i. (m.o.i. = 0.01). Virus titers were determined at different times p.i.as indicated above. Results are shown as means ± SEM (duplicate).

lymphadenitis with lymphoid hyperplasia,focal histiocytosis and moderate infiltration byheterophils.

At day 8 p.i., the injection sites ofwild-type MYXV, MYXV-�M148R and

MYXV-�M148R�M149R showed a differ-ence in the nature of inflammatory infiltratesin the myxomas. With wild-type MYXV(as with the MYXV-�M149R, to a lesserextent), heterophils still composed the main



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cellular population. By contrast, for thelesions associated with the MYXV-�M148Rand MYXV-�M148R�M149R, sequentialinflammatory cellular reactions progressedmore rapidly, as attested by the presence ofinfiltrates with a majority of mononuclearcells (histiocytes and lymphocytes). In theparotid lymph node, a lymphadenitis withlymphoid hyperplasia and focal histiocytosiswas observed for all viruses. As for theprimary site, infiltration by heterophils wasmore pronounced for wild-type MYXV andMYXV-�M149R than for MYXV-�M148Rand MYXV-�M148R�M149R. The presenceof activated fibroblasts with interstitial muci-nosis was greater with wild-type MYXV thanwith all other viruses.

At day 13 p.i., the injection sites ofwild-type MYXV, MYXV-�M148R andMYXV-�M148R�M149R still showed adifference in the nature of inflammatory infil-trates in the myxomas. With wild-type MYXVas with MYXV-�M149R, the proportionsof heterophils and mononuclear cells(histiocytes, lymphocytes, and plasmo-cytes) tended to become counterbalanced. Forthe lesions associated with MYXV-�M148Rand MYXV-�M148R�M149R, mononuclearcells were markedly predominant. The numberof activated fibroblasts and the amount ofinterstitial mucinosis were greater with wild-type MYXV than with all other viruses. In theparotid lymph node, both mutant and wild-type viruses showed a lymphadenitis withlymphoid hyperplasia and focal histiocytosis.

From these observations, we can concludethat the inflammatory reaction is more rapidlyresolved for MYXV-�M148R and MYXV-�M148R�M149R (at day 8 p.i., mononuclearcells were predominant) than for wild-type MYXV and MYXV-�M149R. Anotherdifference was the very high level of cellulardensity of activated fibroblasts and interstitialmucinosis in lesions induced by wild-typeMYXV at day 13 p.i. in comparison withlesions induced by other viruses.

4. DISCUSSION

This study is the first characterization ofM148R and M149R, 2 of the 4 ANK repeat

proteins encoded by MYXV. We demonstratethat M148R and M149R are virulence factorsof MYXV.

Our experiments provide new data onM148R and M149R expression. They werefirst described as early genes coding forcytoplasmic proteins with 9 ANK repeatsand 4 putative transmembrane regions andwith 8 ANK repeats, respectively [5]. Weshow that M148R is expressed as a lategene and that up to 10 ANK repeats can beidentified in the protein. We also show thatM149R is expressed earlier than M148R, ispartially inhibited by AraC and that up to 9ANK repeats can be identified in the protein.The observed decrease in M149R expressionlevel, due to AraC treatment, suggests thatM149R expression level partially relies onviral DNA replication. No sequence clearlycorresponding to early or late promoters wasfound upstream of the ATG of M149R.However, the GG dinucleotide can be foundimmediately after a TAATAT motif (a part ofthe ATG), compatible with an intermediatesignal. On the contrary, an early transcriptiontermination signal is present two nucleotidesupstream of the stop codon. Interestingly, theM148R and M149R form, with MNF, a clusterof genes coding for ANK repeat proteins,temporally expressed in the reverse sense oftheir location, without a transcriptional effectfrom the ones on the others.

M148R and M149R were predicted to becytoplasmic proteins [5]. This was confirmedfor M149R. In contrast we show thatM148R is located in both the cytoplasm andnucleolus, with or without a viral context.This suggests that no viral cofactor is requiredto address M148R to the nucleolus. Thoughno classical nuclear localization signal (NLS)was identified in M148R, it has been shownthat ANK repeats can be used as a nontypical NLS [24]. This has been verified forMNF since deletion of its eighth ANK repeatprevents MNF translocation to the nucleus [6].This may also be the case for M148R.

As initial studies on poxviral ANK repeatproteins, such as Vaccinia virus K1L [3,22] and M-T5 [18], have demonstrated theywere involved in cell tropism or host range



functions, the role of M148R and M149Rin the replication of MYXV was evaluated.We show that deletion of M148R, M149R, orboth, does not induce modification in virusreplication in rabbit cells (fibroblast cell linesor PBMC) or in BGMK cells. Thus M148Rand M149R do not seem to be implicated ineither cell tropism or host range functions,based upon the cell lines examined.

However, we show that single deletion ofM148R or M149R induces a delay in theonset of blepharitis and respiratory infections,an increase of survival time and a dramaticdecrease in mortality rate. Moreover, MYXV-�M148R�M149R infected rabbits expressedonly moderate blepharitis, no signs of respi-ratory infection and totally recovered. Sincethere are no significant differences betweenvirus replication in rabbit lymphocytes, wecan conclude that the observed delay in clin-ical sign onset and the attenuated phenotypecannot be explained by impairment of virusdiffusion after intradermal infection. MYXV-�M149R induced few secondary lesions thatappeared later than with the other virusesand were located on the face only. Sur-prisingly, MYXV-�M148R�M149R infectedrabbits had earlier, smaller and more numer-ous secondary lesions that resorbed faster thanrabbits infected with other viruses, includ-ing the wild type one. Therefore, clinicalsigns observed after infection with MYXV-�M148R�M149R seem to be different fromthe simple addition effect of attenuated phe-notypes induced by each single deleted virus.This suggests that M148R and M149R may actin synergy.

Histological analysis of the primary myx-oma (inoculation site) and parotid lymph nodeshowed that deletion of the M148R geneallowed a quicker resolution of the inflamma-tion. Therefore, like MNF, M148R is locatedin the cell nucleus and seems to play a rolein the subverting of inflammatory response byMYXV. However, deletion of MNF inducesmore drastic attenuation than M148R deletionand they do not seem to be located in the samesubnuclear compartment [6]. Indeed M148Ris located in part in the nucleolus of infectedcells. For many years, the exclusive function

of the nucleolus was thought to be riboso-mal rRNA synthesis and ribosome biogene-sis. However, the nucleolus has been recentlyimplicated in many aspects of cell biologythat include functions such as gene silenc-ing, senescence and cell cycle regulation [19–21]. Numerous viruses encode proteins thatinterfere with the nucleolus, such as EBNA5of the Epstein Barr virus [30], ICP27 of theHerpes simplex virus [16] or Rev and Tat ofHIV-1 [8, 10, 26]. However, reasons for target-ing host-cell nucleoli and mechanisms used tosubvert nucleolar functions are usually specu-lative. It may favor viral transcription, transla-tion or alter the cell cycle in order to promotevirus replication.

If ANK repeat proteins are involved ina large set of functions, the conserved roleof the ANK motif is to be a mediator ofspecific protein-protein interactions. As themajority of ANK repeats poxviral proteins,M148R and M149R have an F-box sequenceat the C-terminus. Both motifs are rare amongviruses but are abundant in poxviruses [2,17]. Moreover, poxviral F-box motifs arenot typical compared to those found ineukaryotes for which F-box motifs are locatedat the N-terminus of the protein and areassociated with WD repeats and leucine-richrepeats as classical protein-protein interactiondomain [12]. F-box proteins are involvedin ubiquitin-dependent proteolysis in cellcycle regulation and signal transduction.They are recognition subunits of ubiquitinligase complexes where the F-box domainbinds to ubiquitin ligase and where protein-protein interaction domain binds the substrateprotein [13]. Based on these observations,Mercer et al. proposed that poxviral ANK-F-box proteins work as described, using ANKrepeats to interact with the target proteinand to direct its ubiquitination [17]. It wasconfirmed for the VACV 68k protein [28]and for five Orf virus proteins [27]. All theseANK-F-box poxviral proteins co-precipitatedwith Skp1 and Cullin-1, two components ofSCF1 ubiquitin ligase complex. All theseproteins have different localizations suchas cytoplasmic, nuclear, perinucleolar, butnone has been shown to be nucleolar as



is M148R. We attempted to co-precipitateCullin-1 in BGMK cells transfected with theGFP fusion of M148R and M149R, underCMV promoter. We succeeded in precipitatingthe fusion proteins, but failed to demonstrateany interaction with neither Cullin-1 norSkp1 (data not shown). These experimentsare not conclusive enough to totally excludean interaction between these proteins. Newexperiments should be done in conditionsof infection, in primate and rabbit cells.Moreover, possible interactions with otherpartners known to interact with some ANK-F-box poxviral proteins such as proteins ofAkt pathway [33] or mainly NF�B signalingpathway, as is the case for K1L [25] andMNF [6], need to be explored.

Acknowledgements. We thank Brigitte Peralta andJosyane Loupias for excellent technical assistance.S. Blanié was supported by funds from the FrenchMinistry of Research and Technology.

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Documents

M148R and M149R are two virulence factors for myxoma virus pathogenesis in the European rabbit